Saliva is the principle protective agent for the mouth and thus is of primary importance to oral health maintenance. Perturbations of salivary secretory mechanisms can consequently lead to serious oral health problems. The objective of this project is to study the membrane and cellular processes that underlie the phenomenon of salivary fluid secretion and thus to contribute to our understanding of the fluid secretory process. Because similar secretory mechanisms are thought to be common to a number of other tissues, this information should be of rather broad applicability and interest. During the present reporting period we have continued our in-depth studies of the function, regulation and molecular biology of the salivary Na-K-2Cl cotransporter. This plasma membrane transport protein is thought to be the major Cl entry pathway into salivary acinar cells and thus to be primarily responsible for driving Cl secretion, and thereby fluid secretion, in salivary glands. Obtaining a better understanding of this protein and its behavior in acinar cells will improve our knowledge of salivary function and dysfunction, as well as possibly providing indications of how to treat the latter. Over the past year we have concentrated on three projects: (i) Characterizing the phosphorylation event associated with upregulation of cotransporter activity in response to beta- adrenergic stimulation. During the present reporting period we have demonstrated that this effect is paralleled by an increase in the number of high affinity binding sites for the cotransporter inhibitor bumetanide in membranes prepared from stimulated acini. We have also shown that the sensitivity of cotransporter fluxes to inhibition by bumetanide is the same in both resting and isoproterenol-stimulated cells, consistent with the hypothesis that beta-adrenergic stimulation, and the accompanying phosphorylation, result in the activation of previously quiescent transporters rather than in a change in the properties of already active proteins. (ii) Mapping the membrane spanning regions of the salivary Na-K-2Cl cotransporter. Working with a theoretical topology scheme derived from hydrophobicity analysis we have been expressing putative membrane spanning regions of the cotransporter in vitro in the presence of canine pancreatic microsomes. The expression vector we have been using appends a peptide containing multiple glycosylation sites onto the C-terminal end of cotransporter sequence being tested. In this way if the test sequence is indeed a membrane spanning region, this peptide will be translocated to the luminal space of the microsome and glycosylated. Our preliminary data using this system only partially confirm the predictions of the theoretical topology scheme. (iii) Use of heterologous expression systems in order to obtain sufficient quantities of functional cotransporter protein for future structure/function studies. In order to obtain sufficient quantities of functional cotransporter protein for structure/function studies in a system which also allows for relatively easy modification of sequence (mutations, truncations, chimeras, etc.) we have recently been investigating the feasibility of expressing this protein in the yeast S. cerevisiae. For these expression studies we added an N-terminal leader to the rat parotid Na-K-2Cl cotransporter sequence. This leader consists of a 9 histidine tag followed by a Factor Xa protease cleavage site. The histidine tag can be used to purify the recombinant cotransporter on a Ni resin and the Factor Xa site will allow us to cleave off the leader sequence near the start methionine of the cotransporter thus essentially leaving uswith the native protein. This construct was ligated into a yeast expression vector incorporating the 5- and 3- untranslated regions of a highly expressed yeast gene, phosphoglyceratekinase. This vector gave expression levelsof histidine tagged cotransporter comparable to salivary tissues, the best mammalian source of this protein identified to date. However, our experiments show that the recombinant protein is significantly less detergent-soluble than the native protein, possibly indicating problems associated with cotransporter trafficking in yeast. We are presently assessing the functional properties of the recombinant protein and attempting to transfer it from yeast membranes to a more suitable lipid environment.